Thiols play a number of important roles in cellular biochemistry. In certain cases they determine protein structure; in others they serve as covalent catalysts; in still others they maintain appropriate oxidation states of proteins and cells. Accordingly, thiol detection and quantitation is important to understanding cellular processes.
Several assays have been developed for thiol quantitation. One such assay is the Thiol and Sulfide Quantitation Kit commercialized by Invitrogen. The kit includes the following: Papain-SSCH3, a disulfide-inhibited papain derivative; L-BAPNA, a chromogenic papain substrate; DTNB (Ellman's reagent) for calibrating the assay; cystamine; L-cysteine, a thiol standard; and a buffer. According to product literature, the chemical basis for the assay involves the following reactions: 1) Papain-SSCH3 is activated in the presence of thiols. 2) Active papain cleaves the substrate L-BAPNA, which releases the p-nitroaniline chromophore. 3) Protein thiols exchange with cystamine to generate 2-mercaptoethylamine, which is detected.
Another thiol quantitation assay has been worked on at Marquette University by Sem and Pullela. See U.S. patent application Ser. No. 11/512,485. Their work focuses on hydroxyl-coumarin-based disulfides, which reportedly react with sulfides present in an assay well. In a related publication the researches noted it was difficult to find a molecule that worked effectively in an assay framework. See, The FASEB Journal, 2008;22:1059.3.
Despite the efforts discussed above, there is still a need for novel thiol quantitation assays that meet the unmet needs of various researchers.